ABSTRACT A decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

中文翻译：

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使用人牙髓间充质干细胞体外胆碱能神经元分化的三种不同方案的比较分析

摘要 胆碱乙酰转移酶（胆碱能神经元中负责乙酰胆碱合成的酶）活性降低会导致神经系统疾病，包括认知能力下降，例如阿尔茨海默病。间充质干细胞 (MSCs) 由于其神经元分化潜力可用作有效的治疗剂。不同来源的MSC在不同的诱导下可能具有不同的分化潜能。已经开发了各种体外方案来将 MSCs 区分为特定的神经元，但使用相同来源的 MSCs 的不同方案的比较效果尚不清楚。为了解决这个问题，牙髓衍生的 MSCs (DPSCs) 使用三种不同的协议分化为胆碱能神经元。在协议 I 中，DPSCs 用含有 1 mM β-巯基乙醇的无血清 ADMEM 预诱导 24 小时，然后用 100 ng/ml 神经生长因子 (NGF) 孵育 6 天。根据协议 II，DPSC 在含有 15 µg/ml D609（tricyclodecan-9-yl-xanthogenate）的无血清 ADMEM 中培养 4 天。根据协议 III，DPSC 在无血清 ADMEM 中培养，其中含有 10 ng/ml 碱性成纤维细胞生长因子 (bFGF)、50 µM 毛喉素、250 ng/ml 音速刺猬 (SHH) 和 0.5 µM 视黄酸（ RA) 7 天。与未处理的细胞相比，DPSCs 在所有协议下都成功转分化，表现出神经元样形态，胆碱能神经元特异性标记物如 ChAT、HB9、ISL1、BETA-3 和 MAP2 在 mRNA 和蛋白质水平上均上调。然而，